Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

Autor: Naoya Tochio, Takehisa Dewa, Takashi Saitoh, Shin-ichi Tate, Masaharu Kondo, Yuichi Togashi, Jun-ichi Uewaki, Takashi Yamamoto, Holger Flechsig, Kohei Umehara, Tetsushi Sakuma
Jazyk: angličtina
Rok vydání: 2016
Předmět:
Zdroj: Scientific Reports
ISSN: 2045-2322
DOI: 10.1038/srep37887
Popis: Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.
Databáze: OpenAIRE
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